The proposal entitled Identification of Antagonists of the Molecular Chaperone Function of CBF? has been written in response to the Program Announcement PA-10-213 entitled Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery (R01). We implement a novel live cell, quenched FRET (FqRET) reporter assay for the molecular interaction of the cellular transcription factor CBF? and the HIV Vif protein in order to discover novel molecular probes for studying the mechanism whereby CBF? binds to and stabilizes Vif. Our target-biased primary assay and the development and optimization of secondary assays and counter screens are planned to identify one or more cell permeable, nontoxic molecular probes that inhibit CBF? binding to Vif. The four Specific Aims will be: (1) Develop and optimize an in-cell quenched FRET (FqRET) assay that will be used in HTS to identify compounds that inhibit the interaction of CBF? with Vif. (2) Develop and optimize orthogonal secondary screens validating that antagonists of CBF? binding to Vif are mechanistically relevant in their ability to reduce the cellular abundance of Vi as well as reduce Vif- dependent degradation of A3G. (3) Counter screen for off-target and cytotoxic hits that affect the ability of CBF? to bind to RUNX1 and function as a cellular transcription factor. And (4) validate selected hits and commercially available chemical analogs for their antiviral activities and target-specificity through functional endpoint assays and bindin studies. At this stage we will have achieved the objective of the PA of having a primary assay and complementary assay systems optimized and validated for transfer and screening at a facility within the NIH MLPCN.